1. Technical Field
The present invention relates to artificial transcription factors capable of artificially regulating gene expression of Escherichia coli by using zinc finger proteins and transcription factors of prokaryote, and engineered E. coli using the same.
2. Background Art
With the advent of post-genome age, researches and analyses are being actively ongoing based on gene information of living things (i.e., organisms). Development and the necessity of a system that may artificially regulate the gene expression draw much attention and are one of fields that are being studied most actively. Up- or down-regulating the expression of specific genes in an organism or a cell and analyzing biological results to be obtained therefrom could allow to discover a function of the specific gene or biological role of the gene. In addition, if the expression of a target gene is properly controlled, this can be utilized as a means for a gene therapy. Furthermore, because the phenotypes of the organism are determined by whether the specific gene is expression or the level of the gene expression in the nature, it can be utilized in development of a desired industry microorganism to prepare an organism expressing with desired phenotypes by regulating the gene expression thereof.
It has been recently reported that it is possible that the gene expression is regulated by using zinc finger proteins. The zinc fingers are known as a DNA binding motif of a DNA binding proteins that are most frequently discovered in eukaryote. The zinc fingers are an active domain that can recognize sequence-specifically a target sequence and can work as transcription repressor by themselves. Furthermore, a new transcription factor can be prepared by fusing the zinc finger proteins with a transcription activating (or suppressing) domain, wherein the zinc finger proteins is used as a DNA binding domain.
It has been recently reported that the target gene expression is able to be up- or down-regulated when a sequence-specific zinc finger DNA binding domain are fused with various types of appropriate effector domains (activating domains or suppressing domains) and expressed in the form of the transcription factor in cells (Liu, Q., Segal, D. J., Ghiara, J. B., and Barb as, C. F., III, 1997 Proc. Natl. Acad, Sci. U.S.A. 94. 5525-5530; Beerli, R. R., Segal, D. J., Dreier, B., and Barbas C. F., III, 1988, Proc. Natl. Acad. Sci. U.S.A. 95, 14628-14633; Beerli R. R., Dreier B. Barbas C.F. 3rd, Positive and negative regulation of endogeneous genes by designed transcription factor, Proc. Natl. Acad. Sci. USA 2000 Feb. 15: 97(4), 1495-500).
However, in the current research for the procaryotic cells, because only the zinc finger domains are used as the transcription factors without the effector domain, only the effect that the gene expression is suppressed can be obtained, which is, however, very weak. Namely, in order to effectively obtain various phenotypes, it is not only necessary to suppress the gene expression but also to activate the expression of various genes.
Therefore, the inventors of the present disclosure has developed novel artificial transcription factors which can up-regulate the gene expression of E. coli as well as down-regulate it by fusing transcription factors of Escherichia coli, which is industrially a useful prokaryote, as the effector domain to a zinc finger domain library, and has discovered that gene expression of E. coli can be artificially regulated by introducing the novel artificial transcription factors in Escherichia coli, and thus engineered E. coli having various phenotypes is able to be prepared through experimentations, thus completing the present invention.